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November 2017 -
Volume 15, Issue 9

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From the Editor

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Original contribution/Clinical Investigation
Diabetes Mellitus - Knowledge, Management and Complications: Survey report from Faisalabad-Pakistan
Ijaz Anwer, Ahmad Shahzad, Kashmira Nanji, Farah Haider, Muhammad Masood Ahmad

Alanine aminotransferase indicates excess weight and dyslipidemia
Mehmet Rami Helvaci, Orhan Ayyildiz* Mustafa Cem Algin, Yusuf Aydin, Abdulrazak Abyad, Lesley Pocock

Comparative Analysis of Antimicrobial Peptides Gene Expression in Susceptible/Resistant Mice Macrophages to Leishmania major Infection

Hamid Daneshvar, Iraj Sharifi, Alireza Kyhani, Amir Tavakoli Kareshk, Arash Asadi

Does socio-economic status of the patients have effect on clinical outcomes after coronary artery bypass grafting surgery?
Forough Razmjooei, Afshin Mansourian, Saeed Kouhpyma

Comparison of the uterine artery Doppler indices during pregnancy between gestational diabetes and diabetes mellitus and healthy pregnant women
Nazanin Farshchian, Farhad Naleini, Amir Masoud Jaafarnejhad,
Parisa Bahrami Kamangar

Survey single dose Gentamicin in treatment of UTI in children with range of 1 month to 13 years old in Jahrom during 2015
Ehsan Rahmanian, Farideh Mogharab,
Vahid Mogharab

Evaluation of control of bleeding by electro cauterization of bleeding points of amplatz sheath tract after percutaneous nephrolithotomy (PCNL) in Jahrom Peymanieh hospital during year 2015-2016
Ali Reza Yousefi , Reza Inaloo

Comparison of the three-finger tracheal palpation technique with triple ID formula to determine endotracheal tube depth in children 2-8 years in 2016-2017
Anahid Maleki, Alireza Ebrahim Soltani, Alireza Takzare, Ebrahim Espahbodi,
Mehrdad Goodarzi , Roya Noori

Effect of Sevoflurane and Propofol on pulmonary arterial pressure during cardiac catheterization in children with congenital heart diseases
Faranak Behnaz, Mahshid Ghasemi , Gholamreza Mohseni, Azim Zaraki
Population and Community Studies

Prevalence and risk factors of obesity in children aged 2-12 years in the Abu Dhabi Islands
Eideh Al-Shehhi, Hessa Al-Dhefairi, Kholoud Abuasi, Noora Al Ali, Mona Al Tunaiji, Ebtihal Darwish

Study and comparison of psychological disorders in normal students and students with multiple sclerosis in Shahrekord
Neda Ardestani-Samani, Mohammad Rabiei, Mohammad Ghasemi-Pirbalooti, Asghar Bayati, Saeid Heidari-Soureshjani

Comparative study of self-concept, physical self-concept, and time perspective between the students with multiple sclerosis and healthy students in Shahrekord
Neda Ardestani-Samani, Mohammad Rabiei, Mohammad Ghasemi-Pirbalooti, Asghar Bayati, Saeid Heidari-Soureshjani

Relationship between Coping Styles and Religious Orientation with Mental Health in the Students of the Nursing-Midwifery Faculty of Zabol
Nasim Dastras, Mohsen Heidari Mokarrar, Majid Dastras, Shirzad Arianmehr

Tuberculosis in Abadan, Iran (2012-2016): An Epidemiological Study
Ali-Asghar ValiPour, Azimeh Karimyan, Mahmood Banarimehr, Marzieh Ghassemi, Maryam Robeyhavi, Rahil Hojjati,
Parvin Gholizadeh

Family Stability and Conflict of Spiritual Beliefs and Superstitions among Yazdi People in Iran: A Qualitative Study
Zahra Pourmovahed , Seyed Saied Mazloomy Mahmoodabad ; Hassan Zareei Mahmoodabadi ; Hossein Tavangar ; Seyed Mojtaba Yassini Ardekani ; Ali Akbar Vaezi

A comparative study of the self-actualization in psychology and Islam
Simin Afrasibi, Zakieh Fattahi

The effectiveness of cognitive - behavioral therapy in reducing the post-traumatic stress symptoms in male students survivors of earthquake in the central district of Varzeghan
Sakineh Salamat, Dr.Ahad Ahangar, Robab Farajzadeh

Reviews

Effects and mechanisms of medicinal plants on stress hormone (cortisol): A systematic review
Kamal Solati, Saeid Heidari-Soureshjani, Lesley Pocock

Comparing Traditional and medical treatments for constipation : A Review Article
Mohammad Yaqub Rajput

A review of anti-measles and anti-rubella antibodies in 15- 25 year old women in Jahrom City in 2011
Ehsan Rahmania , Farideh Mogharab, Vahid Mogharab

Review of percutaneous nephrolithotomy in children below 12 years old in Jahrom hospital, during 2010-2014
Ali Reza Yousefi , Reza Inaloo

Physical and mental health in Islam
Bahador Mehraki, Abdollah Gholami

International Health Affairs

The Challenges of Implementation of Professional Ethics Standards in Clinical Care from the viewpoint of Nursing Students and Nurses
Saeedeh Elhami, Kambiz Saberi, Maryam Ban, Sajedeh Mousaviasl, Nasim Hatefi Moadab, Marzieh Ghassemi

Cognitive Determinants of Physical Activity Intention among Iranian Nurses: An Application of Integrative Model of Behavior Prediction
Arsalan Ghaderi, Firoozeh Mostafavi, Behzad Mahaki, Abdorrahim Afkhamzadeh,
Yadolah Zarezadeh , Erfan Sadeghi

Effect of resilience-based intervention on occupational stress among nurses
Hossein Jafarizadeh, Ebrahim Zhiyani, Nader Aghakhani, Vahid Alinejad, Yaser Moradi

Education and Training

Calculation of Salaries and Benefits of Faculty Members in the Ministry of Health and Medical Education of Iran
Abdolreza Gilavand

The effect of education on self-care behaviors of gastrointestinal side effects on patients undergoing chemotherapy
Shokoh Varaei, Ehsan Abadi Pishe, Shadan Pedram Razie, Lila Nezam Abadi Farahani

Creating and Validating the Faith Inventory for Students at Islamic Azad University of Ahvaz
Solmaz Choheili, Reza Pasha, Gholam Hossein Maktabi, Ehsan Moheb

Creating and Validating the Adjustment Inventory for the Students of Islamic Azad University of Ahvaz
Homa Choheili, Reza Pasha, Gholam Hossein Maktabi, Ehsan Moheb

Evaluating the Quality of Educational Services from the Viewpoints of Radiology Students of Ahvaz Jundishapur University of Medical Sciences
Abdolreza Gilavand, Jafar Fatahiasl

An Investigation of Psychosocial aspect of Iranian Nursing Students' Clinical Setting
Mahsa Boozaripour , Zanyar Karimi, Sima Zohari Anbohi, Amir Almasi-Hashiani, Fariba Borhani

Clinical Research and Methods

Comparison of the Antibacterial Effects of Chlorhexidine Mouth washes with Jaftex Mouth wash on Some Common Oral Microorganisms (An in Vitro Study)
Ebrahim Babadi, Zahra Bamzadeh, Fatemeh Babadi

Study of the effect of plasma jet on Fusarium isolates with ability to produce DON toxins
Elham Galin Abbasian, Mansour Bayat, Arash chaichi Nosrati, Seyed Jamal Hashemi, Mahmood Ghoranneviss

The comparison of anti-inflammatory effect in two methods of topical dexamethasone injection and topical application of ginger alcoholic extract after removing mandibular wisdom teeth
Sahar Zandi, Seyyed Muhammadreza Alavi, Kamran Mirzaie, Ramin Seyedian, Narges Aria, Saman Jokar

The effect of curcumin on growth and adherence of major microorganisms causing tooth decay
Leila Helalat, Ahmad Zarejavid, Alireza Ekrami, Mohammd Hosein Haghighizadeh, Mehdi Shiri Nasab


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November 2017 - Volume 15, Issue 9

Comparative Analysis of Antimicrobial Peptides Gene Expression in Susceptible/Resistant Mice Macrophages to Leishmania major Infection


Hamid Daneshvar
(1)
Iraj Sharifi
(2,3)
Alireza Kyhani
(3)
Amir Tavakoli Kareshk
(2)
Arash Asadi
(2)

(1) Department of Immunology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
(2) Department of Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran
(3) Leishmaniasis Research Center, Kerman University of Medical Sciences, Kerman, Iran


Corresponding author:
Arash Asadi
Department of Parasitology and Mycology,
School of Medicine,
Kerman University of Medical Sciences,
Kerman, Iran

Email:
asadiarash209@yahoo.com

Abstract

Introduction and Objective:
BALB/c and C57BL/6 mouse strains represent immunologically different responses to Leishmania major infection. Antimicrobial peptides (AMPs) for example, cathelicidins and defensins, are unique compounds of innate immunity system with multifunctional effects against invasive pathogens. Nevertheless, they have been less studied in parasitic fields. The aim of the present study was to evaluate the role of AMPs in susceptibility or resistance to L.major infection.

Methodology: Macrophages derived from peritoneal cavity of BALB/c and C57BL/6 mouse strains were exposed to the stationary phase of L. major promastigotes for 3 hours, 24 hours and 7 days. Cell sediments and supernatants from infected (test) and uninfected groups (control) at 3 hours, 24 hours and on 7 days were used for the assessment of infection severity, gene expression of various mouse beta defensins (mBD), Cathelin-related antimicrobial peptide (CRAMP), interleukin (IL)-10, IL-12 and protein assay under standard methods, respectively.

Findings: Based on cytokine profiles evaluated in BALB/c (IL-10, IL-12) and C57BL/6 derived macrophages (IL-10, IL-12), the immunity system was stimulated differently during infection. The inter assay analysis revealed that the test group of BALB/c derived macrophages significantly expressed an up-regulation of CRAMP, mBD1 genes and their related proteins, when they are challenged with L. major parasites. Nevertheless, they also showed infection severity more than those in other strains.

Conclusion: Due to higher expression and release of AMPs by BALB/c derived macrophages, the L. major infection ultimately occurs in BALB/c mice. On the other hand, the release of AMPs is important, but cannot create an absolute protection against leishmania infection.

Key words: Antimicrobial Peptides, Cathelin-Related Antimicrobial Peptide, Murine b-Defensin, Leishmania major, Cytokin


INTRODUCTION

Leishmaniasis is an arthropod-borne disease created by intracellular protozoan parasites of the Leishmania genus (Vega-López, 2012). It is a very important health problem of the recent century in 98 countries and territories (Alvar et al., 2012). The endemic areas of human infections are present mainly in tropics, subtropics, southern Europe and western Asia (Ashford, 1997, Desjeux, 1996). Depending on species and host immunity, several complications such as cutaneous (CL), muccocutaeous (MCL) and visceral leishmaniasis (VL) have been recognized (Herwaldt, 1999). L. major is an ethological agent of CL in different countries such as Iran (Le Blancq et al., 1986, Azizi et al., 2016). Infection is caused, when a female sand-fly inoculates the metacyclic phase of L. major promastigotes into the dermis of a suitable vertebrate host (Dostálová and Volf, 2012). The parasites are engulfed by macrophages for further development (Handman and Bullen, 2002). More interestingly, macrophages involve in both development and killing of parasites. Previous studies have shown that susceptible (BALB/c) and resistant (C57BL/6) mouse strains represent different immune responses to L. major infection (Hejazi et al., 2012, Lazarski et al., 2013, Park et al., 2000). Based on the important role of macrophages, it is necessary to know if new mechanisms such as antimicrobial peptides (AMPs) are employed by them following L. major infection. Historically, the first AMPs was isolated from a soil Bacillus strain and named gramicidin (Dubos, 1939). More than 5,000 AMPs have been identified so far (Zhao et al., 2013). Cathilicidins and defensins are two main groups of AMPs (Ganz, 2003, Lehrer and Ganz, 2002a). Cathelin-related antimicrobial peptide (CRAMP) is the only cathelicidin found in mouse strains and expressed by different kinds of cells or tissues, while a variety of mouse beta defensins (mBD) have been identified (Dorschner et al., 2003, Nizet and Gallo, 2003, Bardan et al., 2004). They kill or inhibit invasion pathogens through direct effects or modulation of inflammatory responses (Deng et al., 2016, Hemshekhar et al., 2016, Chromek et al., 2012, Kovach et al., 2012). Despite being remarkable cases of CL, little study is found about AMPs role in Leishmania infections. The present study aimed to show whether AMPs can affect susceptibility or resistance to L. major infection.

MATERIALS AND METHODS

Ethics approval and consent to participate: To work on animals, we obtained permission number ir.kmu.rec.1394.208 from the ethical board of Kerman University of Medical Science (Kerman, Iran).

Parasite: L. major (strain MRHO/IR/75/ER, Iranian type collection) was purchased from Razi Institute (Karj, Iran) and cultured in 50 ml flask containing RPMI 1640 enriched with 10% heated-inactivated fetal bovine serum (HFBS) and 1% penicillin/streptomycin (pen/strep) antibiotics.

Macrophages Isolation: Macrophages were isolated from BALB/c (n=5) and C57BL/6 (n=5) mouse strains from peritoneal cavity like the previous study (Ray and Dittel, 2010), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) enriched with 10% HFBS and 1% pen/strep antibiotics. The cells were incubated at 37oC in 5% CO2 in humid conditions. For the experiments, the macrophages derived from each strain were separately placed in two sub groups: non-infected (control) and challenged by L. major (test).

Co-incubation of Macrophages with parasite: Macrophage (106 /well) from both strains were separately transferred into 24-wells cell culture plates. Each cell culture plate was designated for one defined time and selected group (5 well for test groups and 5 well for controls). The cells were incubated at 37oC for 6 hours and non adherent cells removed. The stationary phase of L. major promastigotes (10:1) was added only to test groups and incubated at 37oC for 3 hours. The free promastigotes were removed and the cells were incubated at 37oC for an additional 24 hours and 7 days.

Microscopic observation: To measure parasite burden, the test groups were stained using routine Giemsa staining method 3 hours post infection according to a previous study (Faber et al., 2003). Parasite burden (number of parasites per macrophage) and infection rate (% infected macrophages) were obtained by counting the intracellular amastigotes using a light microscope (Nikon, Japan).

Quantitative Real-Time PCR: For analysis of cytokines and AMPs gene expression, whole macrophages from test and control groups were separately harvested at 24 hours post infection with parasites. Total RNA was extracted using RNA Purification kit (Jena Bioscience, Germany) and quantified by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). Three mg was transcribed to complementary DNA (cDNA) using AccuPower®RT PreMix random hexaprimer (Bioneer, Korea). Briefly, 3 µg of RNA was adjusted in 20 µl DEPCI-DW and totally added to each lyophilized tube. Thermal profile was performed as following: 12 cycles (20ºC for 30 seconds, 42ºC for 4 minutes, 55ºC for 30 seconds) and 95ºC for 5 minutes. Quantitative Real-time PCR was utilized using a Rotor GENE Q (Qiagen, Germany). RPII was used to amplify house-keeping cDNA. Other primers were applied to amplify desirable amount of cDNA (Table 1). Briefly, The 15l of each reaction mixture (1ml cDNA, 7 ml SYBR Green, 5ml DW, 1mL primer forward 2.5 Pmol, 1mL primer reverse 2.5 Pmol) was prepared using SYBR Premix EX Taq2 Master Mix (Takara, Japan). Thermal profile was performed as following: 95°C for 1 minute, 40 cycles (94°C for 15 seconds, 58°C for 30 seconds, 72°C for 20 seconds).

ELISA for protein assay: Macrophages were exposed with the stationary phase of L. major promastigotes and kept for 7 days. Due the fact that CRAMP is secreted into cell culture media by macrophages, supernatants were collected for CRAMP assessment by enzyme-linked immunosorbent assay (ELISA). CRAMP assay was accessed using direct ELISA. In this method, the concentration of CRAMP is equal with the absorbance of optical density (OD), and mean of the final OD was calculated as final results. Briefly, a 96-well plate was coated with 5 µg of each supernatant in 50 µl of 0.1M carbonate buffer PH 9.6 and incubated at 4°C FOR 18h. After 3 washes with 300µl of PBS, pH 7.2, 0.1% Tween-20, the plate was then blocked with 100 µl of blocking buffer (PBS, FBS 10%) and incubated at 37°C for 1 hour. Following 3 washes, 100 µl of 1:200 (in PBS, Ph 7.2, 0.1% Tween-20) of horse radish peroxidase conjugated CRAMP antibody (Santacruz, California) was added and incubated for 1 hour at 37º C and washed 3 times at the end of incubation. The plate was incubated with 100 µl of substrate solution for 30 minutes. In the final step, 50µl of stop solution was added and optical density (OD492) detected using ELx800 micro plate reader (BioTek, USA).

FINDINGS

Parasite burden: Initially, we assessed infectivity rate and parasite burden of test groups 3 hours post co-incubation. C57BL/6 derived macrophages had a significant reduction of infection rate (24.5±0.31) as compared to (45±0.73) for BALB/C derived macrophages (Figure 1. A). In the next step, we characterized the parasite burden by counting the number of intracellular amastigotes per macrophage. We saw a significant reduction of parasite burden (2.78±0.10 parasite/macrophage) for C57BL/6 derived macrophages as compared to (8.68±0.22) for other strain (Figure 1. B).

Antimicrobial peptides expression: Real-time PCR was applied to measure the mRNA of defined AMPs following L. major infection. The results were analyzed under CT method. In BALB/c derived macrophages, the test groups expressed all aforementioned genes more than their controls except mBD2, but significantly up-regulation was documented only for CRAMP and mBD1 (Figure 2. A). Unlike the BALB/c macrophages, the test groups of C57BL/6 derived macrophages slightly expressed all mentioned genes except mBD6 more than their controls, but there wasn’t observed any significant differences between them (Figure 2. B). Inter assay analysis showed that the test groups of BALB/c derived macrophages significantly expressed a high level of CRAMP (3.2507±0.0499) and mBD1 (3.0362±0.0701) compared to (0.9852±0.0267) and (1.2074±0.0418) in C57BL/6 derived macrophages, respectively (Figure 2. C ).

Cytokines expression: Cytokines expression was assessed using real-time PCR method, and the findings analyzed under CT method. In BALB/C derived macrophages, the test groups expressed a low level of IL-12 (0.96 ± 0.04) and a significant level of IL-10 (1.91± 0.02) in comparison to (1.005±0.0134) and (1.0016±0.0231) for their controls, respectively (Figure 3. A ). Instead, the test groups of C57BL/6 derived macrophages showed significant expression levels of IL-12 (2.19± 0.05) and a low level of IL-10 (0.85 ± 0.01) versus (1.0036±0.0347) and (1.0021±0.0260) for their controls (Figure 3. B).

Protein assay: Based on AMPs genes expression, the test groups of BALB/c derived macrophages expressed the mRNA levels of CRAMP and mBD1 more than those in C57BL/6. Owing to higher expression of CRAMP compared to mBD1, protein assay was performed only for CRAMP 7 days post infection. Attention to data detected by ELISA method, the test groups of BALB/c derived macrophages released a high level of CRAMP (1.3447±0.010497) in comparison to their controls (0.4706±0.002537) and to (0.4862±0.0021) and (0.4803±0.0022) for the other strain according to the absorbance of optical density assessment (Figure 4).

Click here for Figures 1 A & B

Click here for Figures 2 A, B & C

Click here for Figures 3 A & B

Figure 4

DISCUSSION AND CONCLUSION

Leishmaniasis is a public health problem in many countries (Stefaniak et al., 2002) and there are an estimated 700,000–1 million new cases each year (WHO. Fact sheet. April 2017). It takes a huge economic burden annually. L. major infection is an appropriate model to determine the necessity of immune responses to infection outcome. It has been proven that the increase of some immune effectors, such as IL-12, cause naive lymphocytes differentiate to Th2, which can produce IFN cytokine (Park et al., 2000). This cytokine plays a very important role in activating of macrophage cells. IFN-activated macrophages can destroy the intracellular parasites through a variety of well known mechanisms and induce resistance in C57BL/6 mouse strain (Assreuy et al., 1994). Instead the polarization of Th2 can ultimately predispose BALB/c mice to infection (Chatelain et al., 1992). Similarly to in vivo model, BALB/c and C57BL/6 derived macrophages represent different responses, when they are challenged with L. major parasites (Rabhi et al., 2013). It is possible for genetically different cell types to exhibit an unlike response to the same pathogen like L. major. There is a number of infections referred to as AMPs imbalance, in which they influence susceptibility or resistance to infections (Rivas-Santiago et al., 2009). Surprisingly, these peptides classified in the innate immunity of living organisms, can kill or inhibit pathogens in each category (Lehrer and Ganz, 2002a, Lehrer and Ganz, 2002b, Zasloff, 2002, Bardan et al., 2004, Cavalcante et al., 2017, Kao et al., 2016, Mello et al., 2017, Vieira-Girao et al., 2017). They can be used as new drugs or applied as vaccine and resistance to them is rare (Dabirian et al., 2013, Diamond, 2001, Hancock and Sahl, 2006). The present study aimed to show if these peptides can affect susceptibility or resistance to L. major infection. We designed an in vitro model for studying of AMPs in parasitic infection for the first time. The increased level of infection severity in BALB/c derived macrophages indicates that this type is more sensitive to leishmania infection (Fig1.A-B). According to Sunderkotter et al. (Sunderkötter et al., 1993), C57BL/6 derived macrophages infected by L. major parasites mature faster, which results in the reduction of their infection severity and susceptibility. The results derived from this survey revealed that BALB/c derived macrophages use AMPs especially CRAMP and mBD1more to reduce clinical symptoms (Figure 2. A). It seems that their susceptibility to infection is the immense criteria for the increase of AMPs. There are less in vitro documented studies in this background in the parasitic field. In a described study, human macrophages type expressed a significant up-regulation of CRAMP between the macrophages types (Bank, 2012). Other research has been generally focused on in vivo models. Radzishevsky et al. (Radzishevsky et al., 2005) showed that CRAMP knock-out gene mice represent a severe infectivity rate of L. amozonensis infection in their tissues than wild type. Another aim of this survey was the study of cytokine profiles. Based on the findings related to cytokine profiles, BALB/c derived macrophages expressed a significant up-regulation of IL-10 and a low level of IL-12, while the other type showed completely reverse reaction (Figure 3. A-B). Data from a previous study demonstrated that human derived macrophages type 1 with less sensitivity to L. major infection expressed IL-12 more against other types (Bank, 2012). Finally, the information contained in mRNA molecule must be converted to the synthesis of a new protein. Due to higher expression, the newly synthesized peptide of CRAMP was more measured for BALB/c derived macrophages than the other type (Figure 4). Taken together, AMPs consists of a defense barrier against L. major infection especially in susceptible macrophages, but cannot create an absolute protection following L. major infection.

Acknowledgment
We are grateful from Kerman medical university owing to finance supportive burden of this project and appreciatively the leishmaniasis research center due to use of experience and their requirements.

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